Extraction of anorexigenic and fat-mobilizing substances from animal urine

ABSTRACT

This invention relates to the recovery of anorexigenic and fatmobilizing substances from the urine of normal fasting or coldacclimatized animals.

United States Patent inventors John R. Benton;

James A. F. Stevenson, both of London, Ontario, Canada Appl. No. 460,325

Filed June I, 1965 Patented Nov. 30, 1971 Assignee Canadian Patents andDevelopment Limited Ottawa, Ontario, Canada EXTRACTION OF ANOREXIGENICAND FAT- MOBILIZING SUBSTANCES FROM ANIMAL URINE [561 References CitedUNITED STATES PATENTS 3,002,888 l0/i96l Steifter 167/74 OTHER REFERENCESFunk, C. Further Experiments on the Fat Metabolism Hormone Obtained FromNormal Urine, V. Biol Chem. (May 1933) page X] ill-IV, ScientificProceedings XXVll.

Primary ExaminerAlbert T. Meyers Assistant Examiner-Jerome D. GoldbergAltorneys-Harold A. Weir, Harvey 1. Marshall, Robert A MacRae and JamesA. Lamb ABSTRACT: This invention relates to the recovery of anorexigenicand fat-mobilizing substances from the urine of normal fasting orcold-acclimatized animals.

PATEMEUnuvsomn $624,220

sum 1 or 2 ACID-HYDROLYZED "FAT MOBILIZINO SUBSTANCES" AscENDINO THINLAYER CHROMATOGRAPHY SILICA-ALUMINA PLATE (25M) SAMPLE: 5 1 0. 4%SOLUTION BUTANOL AcETIc ACID WATER (3:1:1) DEVELOPED 2x SPRAY FOLIN'SREAGENT SOLVENT FRONT FMS FMS FMS HUMAN I IA IB FMS AND/OR 8 8 8 8 g $EN Q 9 ELEUCINE O 0 o a 3 TYROSINE o o o u VALINE 0 0 a 0 5 ALANINE o O 0o 6 PROLINE THREONINE a 0 o 0 7 SERIN'E GLUTAMIC ACID GLYCINE 8 8 8 g ifi yCs l i fisPARTlC ACID POINT OF APPLICATION FIGURE i ELECTROPIIORESISON SILICA PLATE (500 44) 720K) VOLTS; 2 HOURS CI'I'RAI'E BUFFER pH 5.820/ 1 5% SOLUTION NINHYDRIN "FAT MOEILIZING SUBSTANCES" LINE OFAPPLICATION FIGURE 3 mzy g/ jzhtudy/a PA7rN dam/r5 3% SOLUTION /NV" 0 aA o (PW/21610. (wad/W1 5 Q1 5w AGE/v73 SHEET 2 [1F 2 UN-HYDROLYZED "FATMOBILIZING SUBSTANCES" SAMPLE: 20 a1 ORMALUEHYDE (:1 :1:10)

SOLVENT FRONT PATENTED NGVBO I97! ASCENDING THIN LAYER CHROMATOGRAPHYSILICA-ETHANOL-PLATE (250 BU'IPANOL-ACETIC ACID-WATER 1% l" DEVELOPED 2xSPRAY NINHYDRIN IN COLLIDINE WAVELENGTH Ill/4 FIGURE /a l nu 6y flzbuy/m S 1 M m 0 F ii m o O T m 51L! w m n M2 M ME I O P m 00 m a 5 m m l EIN ..l mnLU 00a rDlJ 0000 0O 0 w muzfimommw EXTRACTION F ANOREXIGENICAND FAT- MOBILIZING SUBSTANCES FROM ANIMAL URINE The present inventionrelates to the recovery of anorexigenic (appetite-depressing) andfat-mobilizing substances from the urine of normal fasting orcold-acclimatized animals.

More specifically, the present invention relates to the recovery ofanorexigenic and fat-mobilizing substances from the urine of normalfasting or cold-acclimatized rats by a series of extraction steps.

In lancet l, 866 (1958), Lancet 2, 6 (1960) and Endocrinol 69, 648(I961), Chalmers et al. reported the isolation, from the urine offasting man, of a fat-mobilizing substance (FMS) which, when injectedinto mice, caused transient hypoglycemia, ketonemia, hyperlipemia, andincreased mobilization and catabolism of fat with depletion of body fatstores. There was also an increase in blood lipids, including totallipids, phospholipids and free fatty acids. No change in appetite andfood intake accompanied the increased fat catabolism and the decrease inbody weight. in vitro studies with rats showed that minute amounts ofthe active extract were effective in releasing free fatty acids from therat epididymal fat pads. In Lancet 2, 6 (1960) Chalmers et al.postulated that the FMS is polypeptide in nature and that it URINECOLLECTION Adult male rats were routinely maintained for 7 days on ahigh-fat diet in individual metabolism cages. Following this adaptationperiod, all food was removed and urine was collected under toluene forthe next 24 hours. in some experiments, the animals were maintained inan environmental temperature of 5 C. rather than the customary 24 C.

FRACTIONATION The substances FMS-l, 1B (water-soluble) and 1A,(alkaline-soluble) were extracted from combined urine samples by theprocedure depicted as follows:

RAT URINE pH 5.3 alcoholic benzoic acid Benzoic acid precipitate washedwith alcohol Crude FMS-l divided l 1 Crude FMS-1 Crude FMS-1 I 0.5%NazCOa di tilled watcr Alkaline extract i ph 5.3 insoluble 1 sulublvalcohol residue Alcoholic precipitate Crude FMS-1A Crude FMS-ll! 0.5% NaCO; 0.5% NazCO; pll 5.3

alcohol Alkaline extract lyophllized FMS-l Alkaline extract Alcoholicprccipitatv pH 5.3 alcohol Alcoholic precipitate distilled water Aqueousextract lyophilizcd 0.5% NazCOz Alkaline extract FMSAB lyophilizedFMS-1A The procedure for obtaining FMS-l is essentially that of Chalmerset al. and is composed of the following steps: (I) precipitation withalcoholic benzoic acid at pH 5.3; (2) washing with alcohol; (3)extraction of the residue with 0.5 percent sodium carbonate; (4)reprecipitation with alcohol at pH 5.3; (5) reextracting the residuewith 0.5 percent sodium carbonate; (6) drying the supernatant liquid invacuum desiccator to determine the yield of FMS-l; and (7) redissolvingin distilled water for storing at l C. Before bioassaying or injection,all solutions of the extract were adjusted to pH 7.3 with 0.5 percentsodium carbonate. Concentrations were such that each animal received avolume of about 1.0 ml.

Similar purification steps as outlined above for F MS-l were carried outon the crude FMS-1B and FMS-IA materials which were obtained in thefollowing manner: (1) distilled water was added to and mixed with crudeFMS- l and the mixture was filtered; (2) the filtrate containing thecrude FMS-1B was precipitated at pH 5.3 with acid in alcohol solution;and (3) the water-insoluble portion from step 1 containing the crudeFMS-1A was extracted with 0.5 percent sodium carbonate andreprecipitated at pH 5.3 with acid in alcohol solution.

In the above extraction processes it will be understood that the alcoholmay be a lower alcohol such as methanol, ethanol or propanol; that theacid may be an inorganic acid such as hydrochloric or sulfuric acid oran organic acid such as benzoic or acetic acid; and that the alkali maybe an alkali metal or alkaline earth metal hydroxide or carbonate. Itwill also be understood that the acid-alcoholprecipitation steps can becarried out over the broad acid pH range and that the pH of 5.3 isrecited as being the preferred pH for the acid-alcohol system used byapplicants.

Although somewhat variable, the excretion of FMS-1 in the fasting, adultmale rat was found to be approximately -9 mg. per 24 hours.Fractionation of FMSl into 1A and 18 revealed that FMS-l is constitutedof approximately equal parts of 1A and 18 on .a weight basis.

In the following examples, which describe experiments carried out todetermine the chemical nature and mode of action I of FMS-1 IA and 1B,reference is made to drawings in which:

FIG. 1 depicts the results of thin-layer chromatography of amino acidsfollowing the acid hydrolysis of FMS-1, IA and FIG. 2 depicts theresults of thin-layer chromatography of unhydrolyzed FMS-l 1A and 18;

FIG. 3 depicts the results of the electrophoresis of FMS-l; lAandlB,and

FIG. 4 shows the results of the ultraviolet spectral scan of FMS-l, 1Aand 1B.

EXAMPLE I In Vitro Bioassay Samples of about 250 mg. were excised fromepididymal fat pads of male rats, accurately weighed and placed in 50ml. Erlenmeyer flasks containing 5 -ml. of Krebs-Ringer bicarbonatebuffer (pH 7.3-7.4) with 4 percent bovine serum albumen. The increasedrelease of free fatty acids, consequent upon addition of 100 pg. ofextract material, was determined upon incubation for 3 hours at 37 C. ina Dubnoff Metabolic Shaker using air as the gas phase. Themicroprocedure of Dole (J. Clin. Invest. 35, 150 (1960)) was used tomeasure free fatty acids in the medium.

The activities in vitro of these substances, derived from the I samepooled urine samples were 1.45, 1.25, and 2.07 moles free fatty acidsreleased from adipose tissue in 3 hours by 100 pg. of FMS-l, 1A and IB,respectively. It is apparent, therefore, that the order of decreasinglipolytic activity in vitro was 1B FMS-B1 IA. Since FMS-1 is constitutedof approximately equal parts of IA and 13, it would appear that thegreater part of the in vitro lipolytic activity of FMS-1 can beattributed to its content of 1B.

EXAMPLE II Thin-Layer Chromatography The identification of the aminoacids in FMS-1, 1A and 1B was carried out after hydrolysis of 40 mg. in5 ml. of 6N hydrochloric acid and 5 ml. or 88 percent formic acid in asealed glass tube at 100 C. for hours followed by drying under vacuum.Samples of 5p.l. of 0.4 percent aqueous solution were subjected toascending chromatography on a silicaalumina, plate. (250p. thickness)using butanol: acetic acid:

water (3:1: I as solvent. The plates were run twice, dried, and thecolor was developed by spraying with B-napthoquinone-lsodium sulfonatein sodium carbonate solution as described by Niiting(Naturwissenschaften 39, 303 (1952)).

Unhydrolysed material was examined chromatographically by subjecting 201.]. of 3 percent aqueous solutions of FMS-1, 1A and [B to ascendingChromatography on a silica plate (250p. thickness) usingbutanolzethanolzacetic acid: water (4:l:l:10) as solvent. The plateswere run twice, dried and sprayed with ninhydrin in collidine.

As depicted in FIG. 1, FMS-l appears to contain nine to 16 amino acids.An extract from fasting-human urine prepared in the same manner yieldedthe same amino acids upon hydrolysis. A similar pattern of amino acidswas observed in FMS-1A but in hydrolyzed FMS-1B a spot corresponding tovaline was never observed. It is recognized that, if tryptophane werepresent in the original substance, it would probably be destroyed duringthe acid hydrolysis at This destruction can be prevented by hydrolysiswith hydrochloric acid in the presence of Dowex resin and subsequentelution with hydrochloric acid. This was done in one experiment and anadditional spot corresponding to tryptophane was found in bothfractions. Thus, from thin-layer chromatography, it can be tentativelyconcluded that FMS-l and 1A contain 10-17 amino acids (includingtryptophane) whereas 18 contains 9-16 of these, valine being absent.

As shown in FIG. 2, unhydrolyzed FMS-l yields six separate spots onthin-layer chromatography using the plate and solvent described. Four ofthese spots were noted in FMS-IA and three of these spots in FMS-1B.Although the nature of these spots (varying in color from mauve to pinkto yellow) is not known, it is apparent that FMSIA and 1B are chemicallydistinguishable and, further, that FMS-l reveals no spot which cannot befound either in 1A or 1B. In this test it may not be strictly accurateto refer to these substances as unhydrolyzed since the possibility ofsome hydrolysis during chromatography cannot be ignored.

EXAMPLE Ill Amino Acid Identification An examination of acidhydrolysates of FMS-l, IA and 1B was carried out using a TechniconAutoanalyzer. These acid hydrolysates were prepared in the same manneras for thinlayer chromatography.

The following amino acids were identified in FMSl, 1A and 1B: asparticacid, threonine, serine, glutamic acid, proline, glycine, alanine,valine, cystine, isoleucine, leucine, tyrosine, phenylalanine, lysineand histidine. This is in general agreement with the tentativeconclusion drawn from thinlayer chromatographic examination and confirmsthe supposition that complete separation of amino acids was not obtainedwith the thin-layer chromatography. Further, whereas the thin-layerchromatogram failed to reveal valine in FMS-1B, a small amount wasidentified with the Autoanalyzer.

EXAMPLE IV Electrophoresis Combined with thin-layer Chromatography Amixture of silica in 0.1 M citrate buffer (pH 3.8) was spread to athickness of 500p. on a glass plate (8X8 inches). Samples of 20p. 1 of 3percent solutions of FMS-l 1A and 1B in water were placed on the moistplate which was then placed in the horizontal position in anelectrophoresis chamber.

' Citrate buffer (0.1 MzpH 3.8) was employed during electrophoresis for2 hours with a voltage of 200. Following electrophoresis, the plate wasdried and sprayed with ninhydrin reagent for the identification ofspots.

The results of electrophoresis of FMS-l IA and 1B are depicted in FIG.3. Under these conditions, FMS-l revealed six distinctninhydrin-sensitive spots, four of which were evident in IA and theremaining two in 18. The exact nature of these separate substances isnot known at present nor has it been determined whether they occur inFMS-l, 1A and 18 per se or are an artifact created perhaps by partialhydrolysis during the electrophoresis. It is clear however that allninhydrin-sensitive substances in FMS-l can be accounted for by 1A and1B and, further, that the latter two extracts are distinctly different.

CHEMICAL ANALYSIS The results of various chemical analyses of FMS-l, 1Aand 1B are shown in table 1. Attempts to identify the carboydratematerial in these substances using thin-layer chromatography have thusfar been unsuccessful. Attempts to dialyse the material against coldwater have been equally unsuccessful due to possibly the relativeinstability of aqueous solutions of FMS-1, 1A and 18. Activity in vitrocould not be demonstrated in the solution within the dialysing membraneor in the there is a significant loss of activity in 24 hours with nomarked further loss between 24 and 48 hours.

thereafter. A single subcutaneous injection was administered to eachanimal on the 14th morning as follows: A0.5 percent sodium carbonate (pH7.3); B-FMS-l, 2 mg./100 g. body weight; c-FMS-l, 6 mg./l00 g.; D-FMS-l,12 mg./l00 g.; E-FMS-1bA,6 mg./l00 g.;FFMS-1B, 6 mg./100 g.

At the dosage level used, FMS-1 and FMS-1A, but not FMS-1B, caused atransient decrease in food intake and in body weight. This transientdecrease lasted for 1-2 days. It is of interest that FMS-1B, which hasthe greatest lipolytic activity in vitro, had no effect upon either foodintake or body weight. The mean decreases of food intake and body weight24 hours after injection of FMS-l or FMS-1A were all statisticallysignificant, (P 0.01).

Using the three dosage levels of FMS-l, 2, 6 and 12 mg./ 100 g. bodyweight, an approximate proportionality between dose and degree ofdecrease of food intake and body weight was observed on day 1. Of thesedecreases, only that following 2 mg./ 100 g. was not statisticallysignificant; the decreases brought about by 6 and 12 mg./l00 dosagelevels were significant (p 0.02 for body weight decreases and 0.05 forfood intake decreases).

SOLUBLE) AND FMS-1B (NEUTRAL WATER SOLUBLE) Percent Total Total Carbo-Hexos- Cholns- Phos- Pentose Fraction nitrogen hydrate amine terolphorus Biuret color test FMS-1.." 8. 4 1. 25 0.31 2. 3 1.1 Pink-blue...Negative. FMS-1A.. 10.0 1. 00 0.24 4. 1 1.0 Blue-mauve. Do. FMS1B 3. 80. 69 0.23 0. 75 1. 8 Bluepink." Do.

TABLE II EXAMPLE V] In vitro activity changes in solutions of FMS-1,FMS-1A and Fat Mobilization in Vivo FMS-1B stored at 5 C. in stopperedtest tubes Fraction 0 hours Z: initial activity initial pH 24 hours 48hours of solution FMS-l I00 23 II 9.3 FMS-1A I00 50 48 9.8 FMS-1B I00 6868 3.1

' solution in aqueous sodium carbonate solution in distilled water.

EXAMPLE V Food Intake and Body Weight Thirty-five rats of the Wistarstrain weighing 260-300 g. were divided into seven groups of five eachand were provided with a high-fat diet for 13 days prior to injectionand 6 days after injection. Food intakes were measured daily; bodyweights were measured three times before injection and dailyTwenty-seven rats of the Sprague-Dawley strain weighing 400-500 g. weredivided into four groups of six and one group of three animals. Extractswere injected at a level of 6 mg./l00 g. body weight as follows: A-0.5percent sodium carbonate (pH 7.3); B-FMS-l; CFMS-lA; DFMS-1B; E-HypoxFMS" from urine of fasted hypophysectomized rats. Six hours afterinjection, the animals were anesthetized by the intraperitonealinjections of sodium pentobarbital (6 mg./ g. body weight) and bloodsamples were obtained by cardiac puncture. About 2 g. of liver wererapidly removed, accurately weighted and digested in potassium hydroxidesolution for the determination of total crude fatty acids by a modifiedLibermann procedure. On blood samples the following were measured:serum-free fatty acids, plasma total cholesterol and blood ketones.

The results of blood and liver analyses 6 hours after injection ofFMS-1, 1A, 1B and l-lypox FMS" are shown in table Ill. The extractsFMS-l and FMS-1B caused significant increases in the level of serum-freefatty acids and in the concentration of total crude fatty acids in theliver. In addition, FMS-1B caused a significant increase in the bloodlevel of ketones. FMS 1A and Hypox FMS" were without effect upon any ofthese parameters, nor did any of the extracts significantly alter theplasma concentration of cholesterol. It would seem therefore, that, invivo, FMS-1A and Hypox FMS have no fat-mobilizing properties and,further, this property of F MS-l may be attributable to its content ofFMS-1B.

TABLE Ill-THE EFFECT OF PRIMARY EXTRACTS ON FAT MOBILIZA- TION IN VIVOIN THE RAT (Results expressed as mean :t: S.E.M.)

1 P 0.05 compared with control value.

EXAMPLE Vll EXAMPLE IX Blood Sugar Levels Excretion of Hypox FMS" byFasting, Hypophysectomized Twenty rats of the Sprague-Dawley strainweighing 400-450 g. were divided into four groups of five each. After 5Thirteen hypophysectomized rats of the wistal' Snail all food wasremoved, the animals were injected with 0.5 perg g 220-250 8- housed inindividual metabolism cent sodium carbonate (pH 7.3) or with one of theextracts cages and Provided with the high-fat for a Period 10 FMS-lFMS-1A and FMS-1B at a l l f 2 /100 days. At the end of this time, sixrats were rejected on the basis Blood samples were taken from the tip ofthe tail just before of either excessive Weight 1055 fats) Weight gainsinjection and at L5 and 3 hours post injection. Blood sugar greater than1 8- P y (four The remaining Seven was determined b a miero difi i f h Ns animals were fasted for 24 hours during which urine was colgyiprocedure, lected. At the termination of the experiment, brain and ad-The blood sugar level wa i ifi l decreased 1 5 and 3 jacent tissues weredissected out, the seela turcica was exhours after the animals wereinjected with FMS-l, 1A or 18 as mined in the gross and the mpl en f pii ry shown in table lV. l5 gland was confirmed. An extract was preparedfrom the pooled urine sample as previously described.

Fasting, hypophysectomized rats excreted a material in TABLEW urinewhich on extraction yielded 13.0 mg./rat/24 hours. In

vitro assay of this material failed to reveal any lipolytic activi-Effect of urine extracts on blood sugar levels in rats of urine ty.Further, examination by thin-layer chromatography sug- (Resultsexpressed as the mean::S.E.M. for 5 rats) gested that this materialdiffered from the FMS-l extracted from the urine of fasting, intactrats.

In summary, applicants have isolated from the urine of fast- Blood su arsum! 0 hum s ing or cold-acclimatized rats an-anorexigenic andfat-mobiliz- 25 ing substance which can be further fractionated into twofracgiii'g: 3 tions, the first of which being water-soluble and thesecond of FMS-IA szgslss 65;2:13 5 which being water-insoluble butalkaline-soluble. Experimen- PMs-1B 7820.86 6413.06 5413.66 tal resultsindicate that the fat-mobilizing properties are associated with thewater-soluble fraction, while the anorexigenic properties are associatedwith the water-insoluble, alkaline-soluble fraction. Both fractionsappear to cause Rom hypoglycemia.

Although the anorexigenic and fat-mobilizing substances of P 0.02compared with O-hour value ofthe same group -i+P 0.00l

the present invention have been obtained from the urine of normalfasting or cold-acclimatized rats, it is believed that similarsubstances with similar physiological properties could EXAMPLE beobtained from the urine of other fasting or cold-accli- Excretion ofFMS-l by Non-fasting, Cold-exposed Rats matized forms of animal lifesuch as horses, cows, goats,

Twelve rats of the Sprague-Dawley strain weighing approxi. 40 sheep.dogs, rabbits, and the like. mately 300 g. were maintained in metabolismcages on the we claim: high-fat diet for 7 days at 24 C. On the 7th day,urine was col- A method of extracting anorexigenic and fai'mobililinfipolypeptide-containing substances from the urine of rats which comprisessubjecting said rats to stress by fasting or cold-acclimatization;collecting urine from the stressed rats; precipitating, at a pH of 5.3,said substances in crude form from said urine by the addition ofethanol; and extracting this crude product, first with water and thenwith aqueous sodium carbonate, to obtain a water-soluble lected and anextract prepared. The animals were then transferred to the cold roomat'5 C. and urine was collected daily for 3 days and then pooled. Fromthis 3-day pooled sample. an 45 extract was prepared. The activities ofthe extracts were measured in vitro by the fat-pad assay.

The urine collected from the rats fed at an environmental temperature of24 C. when extracted for FMS-1 yielded 7.7 mg./rat/24 hours, but thisshowed no lipolytic activity upon assay in vitro. From the urinecollected from the same rats in l M the fed state in a cold environmentof 5 C. only 1.7 mg./rat/24 2352;2 :3 2 gf i f g gi it z gg so u efracuon hours of material was obtained but it had an activity of aboutAn anorexigenic, polypeptidemomainingi watepinsolw one-thud h P F offasted 5 ble, alkaline-soluble fraction of the product prepared by therats. Examination of this material by thin-layer chromatogprocess d fidi claim raphy indicated a close similarity, if not identity, withFMS-l.

2. An anorexigenic, polypeptide-containing, water-insoluble,alkaline-soluble fraction of the product prepared by the process definedin claim 1.